A Study of the Roles of Selected Arginine and Lysine Residues of TAFI in Its Activation to TAFIa by the Thrombin-Thrombomodulin Complex

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Abstract

Thrombin-activatable Fibrinolysis Inhibitor (TAFI) is a 60 kDa plasma protein that can be activated to the enzyme, TAFIa, by thrombin, plasmin or trypsin. TAFIa is a carboxypeptidase B-like enzyme that attenuates fibrinolysis. Thrombomodulin (TM) is a cofactor which increases the overall efficiency of thrombin-mediated TAFI activation by 1250-fold. Thus, the thrombin-TM complex is believed to be the physiological TAFI activator.
The minimal structure of TM required for efficient TAFI activation contains the EGF-like domains 3 through 6. New structure models have postulated that the C-loop of TM EGF-like domain 3 has a negatively charged molecular surface that could interact with several positively charged surface patches on TAFI.
In this study, we constructed recombinant TAFI variants to assess whether the selected positively charged residues on TAFI complement the negative electrostatic potential of the TM EGF-like domain 3, thereby promoting the TAFI-TM interaction in the formation of the ternary thrombin/TM/TAFI complex.
TAFI has exclusive triple lysine residues on its activation peptide. When they are substituted by alanine residues (K42/43/44A), compared to the wild-type, the catalytic efficiencies for TAFI activation by thrombin in the presence and absence of TM decreased by factors of 9 and 3.5, respectively. Other derivatives of TAFI with alanine point mutations at positions K133, K211, K212, and R220, which together represent one positively charged surface patch of TAFI, showed decreased catalytic efficiencies for TAFI activation by thrombin-TM complex from 2.4 to 2.9-fold. A second positive surface patch includes residues K240 and R275. Alanine mutations of these two residues caused decreased catalytic efficiencies by 1.7 and 2.1-fold, respectively. Together, our data show that no single mutation completely eliminates TM dependence in TAFI activation by thrombin, but each mutated residue contributes in the formation of the ternary thrombin/TM/TAFI complex. In addition, all TAFIa derivatives had half lives (8.1 ± 0.6 min) comparable to that of wild-type TAFIa (8.4 ± 0.3 min) at 37 ºC, suggesting that these residues are not involved in TAFIa inactivation by conformational instability.